Protocol For Normalisation Of Mrna Samples?

Question

[14134125465346445]

Is there an easy, inexpensive, not too labor intensive way to normalise mRNA samples so that even though one loses information of gene expression levels, each of the transcripts in the transcriptome is equally represented in the sample for sequencing? This is to say, one has a uniform distribution of transcripts, so that the lowly-expressed transcripts still have a good chance of being sequenced.

I have seen a few papers mentioning protocols for mRNA normalization, but I can't tell if any of them is practical in real life wet lab situations.

[Malachi Griffith]

This might be more of a SeqAnswers question but perhaps you could post links to the papers or specific protocols you mention...

[Steve Lianoglou]

Can you update your question with links to some of the references for mRNA normalization that you are alluding to? Aside from ribosomal RNA depletion, I've never really heard of people trying to do this before. There's also the stuff that Gingeras has done, which is to purify and sequence RNA from different cellular compartments, but this isn't really what you're suggesting either.

Answer 1

[Malachi Griffith]

There is some talk of normalization by use of a duplex specific nuclease (DSN). This has been discussed in several threads on SeqAnswers here. Another option is to take your cDNA library, capture it with a capture reagent (e.g. exome array or liquid capture kit), and then sequence it. This will reduce the representation of highly expressed transcripts in your library and allow your library depth to be spread more evenly across the transcriptome.

[14134125465346445]

I take you would need to know the tags to capture if you wanted to reduce the representation of highly expressed transcripts, right?

Answer 2

[klaugo]

Have you looked at Springer protocols: Accessing the transcriptome: how to normalize mRNA pools ?

http://www.ncbi.nlm.nih.gov/pubmed/22065434

Answer 3

[14134125465346445]

Thanks @Malachig for your DSN link. I found the link to the commercial product by Evrogen here:
http://www.evrogen.com/technologies/normalization.shtml

They claim their method is compatible with nextgen sequencing platforms:

cDNA normalization using duplex-specific nuclease (DSN) is a highly efficient approach that can be applied for normalization of full-length-enriched cDNA (Zhulidov et al., 2004; Zhulidov et al., 2005). The resulting cDNA contains equalized abundance of different transcripts and can be used for construction of cDNA libraries and for direct sequencing, including high-throughput sequencing on the next generation sequencing platforms (Roche/454, ABI/SOLiD or Illumina/Solexa).

And the most up-to-date reference seems to be this Curr Protoc Mol Biol. paper by Bogdanova et al.:
http://www.ncbi.nlm.nih.gov/pubmed/20373503